Myosin is a family of proteins that demonstrate force-generated ATPase activity when it interacts with actin. Myosin heavy chain (MHC) protein isoform diversity in vertebrates is generated by multiple genes as well as by alternative splicing of the pre-mRNA. Previous studies in this laboratory have demonstrated the existence of a neuron-specific nonmuscle MHC-B isoform, in addition to the ubiquitously expressed form of nonmuscle MHC-B. This neuron-specific isoform is generated by alternative splicing of a single cassette type of exon N30. Inclusion or exclusion of exon N30 is cell type-specific, with inclusion being restricted to neuronal cells and being regulated during cell differentiation. To study the mechanism(s) of neuron-specific alternative splicing, we first established a minigene transient transfection system. A minigene, which contains the alternative exon N30 and flanking exons and introns, was constructed and was transfected into a number of cell lines including human neuronal retinoblastoma Y79 cells. Following transfection of this minigene, the mRNA derived from the minigene construct were analyzed by RT-PCR. The transfected minigene reproduced cell type-specific and differentiation-dependent alternative splicing of the N30 exon. Inclusion of the N30 exon in the mRNA from the transfected minigene occurs in differentiated Y79 cells that have been treated with butyrate, but not in the undifferentiated Y79 cells and non-neuronal cell lines. Systematic deletion and mutation analysis of the minigene construct established that neuron-specific N30 exon recognition requires an intronic cis-acting RNA sequence located approximately 1.5 kb downstream of the N30 exon. A 19 nucleotide sequence (UGCAUGUCGUACUGCAUGU) in this region was determined to be critical for its cis-regulatory function.